Many vitally important proteins which are secreted (such as immunoglobulins and polypeptide hormones) or present at the cell surface (including cell adhesion molecules and signaling receptors) are produced and folded by the endoplasmic reticulum (ER). If the ER has problems folding these proteins, a compensatory ER stress response (a.k.a. Unfolded Protein Response) is triggered to enhance ER-related folding processes. ER-produced proteins are frequently N-glycosylated with asparagine-bound sugar polymers (glycans). The glycans are intimately involved in folding of N-glycoproteins, and can also participate in their functions after secretion or reaching the cell surface. The ER-associated lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol provides the glycan (Glc3Man9GlcNAc2) used to make N-glycoproteins. Many key features of LLO synthesis have been known for over 20 years, but functional acute regulation of the pathway has been poorly understood. This research proposal focuses on the P.I.'s discovery of LLO synthesis regulation by the ER stress response, mediating a feedback loop which can compensate when LLO insufficiency is the original cause of ER dysfunction. This involves both a LLO biosynthetic arm and a counter-intuitive LLO degradative arm. Thus LLO production is not hard-wired, but instead is constantly monitored and adjusted. Aim I will elucidate the mechanism by which the ER stress response controls one component of the LLO biosynthetic arm, elevation of levels of nucleotide-sugars which are the precursors of glycans. This Aim will: determine how stress-induced hexose phosphates elevate nucleotide-sugar pools; identify steps in LLO synthesis responsive to nucleotide-sugar control; and explore the stress signal transducer and effector involved. Aim II will provide important new information about an unexpected activity of mannose- 6-phosphate (M6P) and its key role in the LLO degradative arm. The P.I. discovered that M6P is elevated by ER stress, and causes release of glycan from Glc3Man9GlcNAc2-P-P-dolichol. This Aim will: develop mimics and antagonists of M6P to elucidate its cellular action; explore the role of M6P-released glycans in ER homeostasis; and test the hypothesis that the degradative arm represents a novel host defense mechanism against viral envelope N-glycoprotein synthesis (a process using LLO and likely to induce ER stress) with herpes simplex-1 as a model. The clinical relevance is two-fold. First, there are 13 human genetic diseases in the family Congenital Disorders of Glycosylation (CDG) Type I, with defective LLO synthesis. CDG-I cells have ER dysfunction due to poor N-glycosylation, and patients have many clinical difficulties. Fundamental new insights into the regulatory systems which may impact CDG-I will be gained from this work. Second, this work will generate new information about the ER stress response, which governs the productivity of the ER, is essential for the secretory functions of plasma cells (immunoglobulins) and pancreatic islets (insulin), and when aberrantly controlled can cause neurological diseases, cholesterol imbalance, and obesity. Public Health Relevance: The public health relevance of this research is three-fold. First, new strategies for treating human diseases in which patients have abnormal carbohydrate attachment to protein will be evaluated. Second, this work will generate new information about the how cells respond to stress, which is an important factor in the production of immunoglobulins to fight infection, in the production of insulin to prevent diabetes, and in diseases involving the nervous system, cholesterol, and obesity. Third, we will evaluate a potential host- defense mechanism against certain infectious human viruses.